This invention relates in general to hydrazidyl, bis-hydrazidyl and bis-aminomethyl carbonyl protease inhibitors, particularly such inhibitors of cysteine and serine proteases, more particularly compounds which inhibit cysteine proteases, even more particularly compounds which inhibit cysteine proteases of the papain superfamily, yet more particularly compounds which inhibit cysteine proteases of the cathepsin family, most particularly compounds which inhibit cathepsin K. Such compounds are particularly useful for treating diseases in which cysteine proteases are implicated, especially diseases of excessive bone or cartilage loss, e.g., osteoporosis, periodontitis, and arthritis.
Cathepsins are a family of enzymes which are part of the papain superfamily of cysteine proteases. Cathepsins B, H, L, N and S have been described in the literature. Recently, cathepsin K polypeptide and the cDNA encoding such polypeptide were disclosed in U.S. Pat. No. 5,501,969 (called cathepsin O therein). Cathepsin K has been recently expressed, purified, and characterized. Bossard, M. J., et al., (1996) J. Biol. Chem. 271, 12517-12524; Drake, F. H., et al., (1996) J. Biol. Chem. 271, 12511-12516; Bromme, D., et al., (1996) J. Biol. Chem. 271, 2126-2132.
Cathepsin K has been variously denoted as cathepsin O or cathepsin O2 in the literature. The designation cathepsin K is considered to be the more appropriate one.
Cathepsins function in the normal physiological process of protein degradation in animals, including humans, e.g., in the degradation of connective tissue. However, elevated levels of these enzymes in the body can result in pathological conditions leading to disease. Thus, cathepsins have been implicated as causative agents in various disease states, including but not limited to, infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei brucei, and Crithidia fusiculata; as well as in schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like. See International Publication Number WO 94/04172, published on Mar. 3, 1994, and references cited therein. See also European Patent Application EP 0 603 873 A1, and references cited therein. Two bacterial cysteine proteases from P. gingivallis, called gingipains, have been implicated in the pathogenesis of gingivitis. Potempa, J., et al. (1994) Perspectives in Drug Discovery and Design, 2, 445-458.
Cathepsin K is believed to play a causative role in diseases of excessive bone or cartilage loss. Bone is composed of a protein matrix in which spindle- or plate-shaped crystals of hydroxyapatite are incorporated. Type I collagen represents the major structural protein of bone comprising approximately 90% of the protein matrix. The remaining 10% of matrix is composed of a number of non-collagenous proteins, including osteocalcin, proteoglycans, osteopontin, osteonectin, thrombospondin, fibronectin, and bone sialoprotein. Skeletal bone undergoes remodelling at discrete foci throughout life. These foci, or remodelling units, undergo a cycle consisting of a bone resorption phase followed by a phase of bone replacement.
Bone resorption is carried out by osteoclasts, which are multinuclear cells of hematopoietic lineage. The osteoclasts adhere to the bone surface and form a tight sealing zone, followed by extensive membrane ruffling on their apical (i.e., resorbing) surface. This creates an enclosed extracellular compartment on the bone surface that is acidified by proton pumps in the ruffled membrane, and into which the osteoclast secretes proteolytic enzymes. The low pH of the compartment dissolves hydroxyapatite crystals at the bone surface, while the proteolytic enzymes digest the protein matrix. In this way, a resorption lacuna, or pit, is formed. At the end of this phase of the cycle, osteoblasts lay down a new protein matrix that is subsequently mineralized. In several disease states, such as osteoporosis and Paget""s disease, the normal balance between bone resorption and formation is disrupted, and there is a net loss of bone at each cycle. Ultimately, this leads to weakening of the bone and may result in increased fracture risk with minimal trauma.
Several published studies have demonstrated that inhibitors of cysteine proteases are effective at inhibiting osteoclast-mediated bone resorption, and indicate an essential role for a cysteine proteases in bone resorption. For example, Delaisse, et al., Biochem. J., 1980, 192, 365, disclose a series of protease inhibitors in a mouse bone organ culture system and suggest that inhibitors of cysteine proteases (e.g., leupeptin, Z-Phe-Ala-CHN2) prevent bone resorption, while serine protease inhibitors were ineffective. Delaisse, et al., Biochem. Biophys. Res. Commun., 1984, 125, 441, disclose that E-64 and leupeptin are also effective at preventing bone resorption in vivo, as measured by acute changes in serum calcium in rats on calcium deficient diets. Lerner, et al., J. Bone Min. Res., 1992, 7, 433, disclose that cystatin, an endogenous cysteine protease inhibitor, inhibits PFH stimulated bone resorption in mouse calvariae. Other studies, such as by Delaisse, et al., Bone, 1987, 8, 305, Hill, et al., J. Cell. Biochem., 1994, 56, 118, and Everts, et al., J. Cell. Physiol., 1992, 150, 221, also report a correlation between inhibition of cysteine protease activity and bone resorption. Tezuka, et al., J. Biol. Chem., 1994, 269, 1106, Inaoka, et al., Biochem. Biophys. Res. Commun., 1995, 206, 89 and Shi, et al., FEBS Lett., 1995, 357, 129 disclose that under normal conditions cathepsin K, a cysteine protease, is abundantly expressed in osteoclasts and may be the major cysteine protease present in these cells.
The abundant selective expression of cathepsin K in osteoclasts strongly suggests that this enzyme is essential for bone resorption. Thus, selective inhibition of cathepsin K may provide an effective treatment for diseases of excessive bone loss, including, but not limited to, osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget""s disease, hypercalcemia of malignancy, and metabolic bone disease. Cathepsin K levels have also been demonstrated to be elevated in chondroclasts of osteoarthritic synovium. Thus, selective inhibition of cathepsin K may also be useful for treating diseases of excessive cartilage or matrix degradation, including, but not limited to, osteoarthritis and rheumatoid arthritis. Metastatic neoplastic cells also typically express high levels of proteolytic enzymes that degrade the surrounding matrix. Thus, selective inhibition of cathepsin K may also be useful for treating certain neoplastic diseases.
Several cysteine protease inhibitors are known. Palmer, (1995) J. Med. Chem., 38, 3193, disclose certain vinyl sulfones which irreversibly inhibit cysteine proteases, such as the cathepsins B, L, S, O2 and cruzain. Other classes of compounds, such as aldehydes, nitrites, xcex1-ketocarbonyl compounds, halomethyl ketones, diazomethyl ketones, (acyloxy)methyl ketones, ketomethylsulfonium salts and epoxy succinyl compounds have also been reported to inhibit cysteine proteases. See Palmer, id, and references cited therein.
U.S. Pat. No. 4,518,528 discloses peptidyl fluoromethyl ketones as irreversible inhibitors of cysteine protease. Published International Patent Application No. WO 94/04172, and European Patent Application Nos. EP 0 525 420 A1, EP 0 603 873 A1, and EP 0 611 756 A2 describe alkoxymethyl and mercaptomethyl ketones which inhibit the cysteine proteases cathepsins B, H and L. International Patent Application No. PCT/US94/08868 and and European Patent Application No. EP 0 623 592 A1 describe alkoxymethyl and mercaptomethyl ketones which inhibit the cysteine protease IL-1xcex2 convertase. Alkoxymethyl and mercaptomethyl ketones have also been described as inhibitors of the serine protease kininogenase (International Patent Application No. PCT/GB91/01479).
Azapeptides which are designed to deliver the azaamino acid to the active site of serine proteases, and which possess a good leaving group, are disclosed by Elmore et al., Biochem. J., 1968, 107, 103, Garker et al., Biochem. J., 1974, 139, 555, Gray et al., Tetrahedron, 1977, 33, 837, Gupton et al., J. Biol. Chem., 1984, 259, 4279, Powers et al., J. Biol. Chem., 1984, 259, 4288, and are known to inhibit serine proteases. In addition, J. Med. Chem., 1992, 35, 4279, discloses certain azapeptide esters as cysteine protease inhibitors.
Antipain and leupeptin are described as reversible inhibitors of cysteine protease in McConnell et al., J. Med. Chem., 33, 86; and also have been disclosed as inhibitors of serine protease in Umezawa et al., 45 Meth. Enzymol. 678. E64 and its synthetic analogs are also well-known cysteine protease inhibitors (Barrett, Biochem. J., 201, 189, and Grinde, Biochem. Biophys. Acta, 701, 328). U.S. Pat. No. 5,142,056 describes 1,3-diamido-propanones which inhibit HIV protease. 1,3-diamido-propanones have also been described as analgesic agents (U.S. Pat. Nos. 4,749,792 and 4,638,010).
Certain heterocyclic derivatives of amino acids have been disclosed in the art. For instance, Hamada, et al., PEPTIDE CHEMISTRY, 1983. Proceedings of the 21st Symposium on Peptide Chemistry (1984), and Boden, et al., Tet. Lett., 1994, 35, 8271 (1994) disclose thiazole derivatives; and Borg, et al., 1995, 60, 3112, disclose oxadiazole and triazole derivatives.
The synthesis of azatides (polyacylhydrazides) as peptide mimetics has recently been disclosed by Han and Janda, J. Am. Chem. Soc. 1996, 118, 2539.
Thus, a structurally diverse variety of cysteine protease inhibitors have been identified. However, these known inhibitors are not considered suitable for use as therapeutic agents in animals, especially humans, because they suffer from various shortcomings. These shortcomings include lack of selectivity, cytotoxicity, poor solubility, and overly rapid plasma clearance. A need therefore exists for methods of treating diseases caused by pathological levels of cysteine proteases, including cathepsins, especially cathepsin K, and for novel inhibitor compounds useful in such methods.
We have now discovered a novel class of hydrazidyl, bis-hydrazidyl and bis-aminomethyl carbonyl compounds which are protease inhibitors, most particularly of cathepsin K.
An object of the present invention is to provide hydrazidyl, bis-hydrazidyl and bis-aminomethyl carbonyl protease inhibitors, particularly such inhibitors of cysteine and serine proteases, more particularly such compounds which inhibit cysteine proteases, even more particularly such compounds which inhibit cysteine proteases of the papain superfamily, yet more particularly such compounds which inhibit cysteine proteases of the cathepsin family, most particularly such compounds which inhibit cathepsin K, and which are useful for treating diseases which may be therapeutically modified by altering the activity of such proteases.
Accordingly, in the first aspect, this invention provides a compound according to Formula I.
In another aspect, this invention provides a pharmaceutical composition comprising a compound according to Formula I and a pharmaceutically acceptable carrier, diluent or excipient.
In yet another aspect, this invention provides a method of treating diseases in which the disease pathology may be therapeutically modified by inhibiting proteases, particularly cysteine and serine proteases, more particularly cysteine proteases, even more particularly cysteine proteases of the papain superfamily, yet more particularly cysteine proteases of the cathepsin family, most particularly cathepsin K.
In a particular aspect, the compounds of this invention are especially useful for treating diseases characterized by bone loss, such as osteoporosis and gingival diseases, such as gingivitis and periodontitis, or by excessive cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis.
The present invention provides compounds of Formula I: 
L=C2-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl, CH(R66)NR60R68, CH(R66)Ar, CH(R66)OArxe2x80x2, NR66R67;
M=C(O), SO2;
G=
J=C(O), SO2;
T=Ar, Het;
V=C3-7cycloalkyl;
Wxe2x95x90H, xe2x80x94CN, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94COR7, xe2x80x94CO2R6, xe2x80x94CONHR6, xe2x80x94SO2NHR6, xe2x80x94NHSO2R6, xe2x80x94NHCOR7, xe2x80x94Oxe2x80x94COR6, xe2x80x94SR6, NRxe2x80x2R6, NRxe2x80x2(Cxe2x95x90NH)NHR5, Cl, Br, I, F;
Xxe2x95x90Yxe2x95x90Zxe2x95x90N, O, S or CR4,
xe2x80x83provided that at least two of X, Y and Z are heteroatoms and at least one of X, Y and Z is N, or one of X, Y and Z is Cxe2x95x90N, Cxe2x95x90C or Nxe2x95x90N and the other two are CR4 or N, provided that X, Y and Z together comprise at least two N;
indicates a single or double bond in the five-membered heterocycle;
m=0, 1, 2;
n=1 to 6;
xcfx86=0, 1, 2;
Ar=phenyl, naphthyl, optionally substituted by one or more of Ph-C0-6alkyl, Het-C0-6alkyl, C1-6alkoxy, Ph-C0-6alkoxy, Het-C0-6alkoxy, OH, (CH2)1-6NR58R59, O(CH2)1-6NR58R59;
Arxe2x80x2=phenyl or naphthyl, optionally substituted by one or more of Ph-C0-6alkyl, Het-C0-6alkyl, C1-6alkoxy, Ph-C0-6alkoxy, Het-C0-6alkoxy, OH, (CH2)1-6NR58R59, O(CH2)1-6NR58R59, or halogen;
Rxe2x80x2=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R1=H, C1-6alkyl;
R2=C4-6alkyl, C4-6alkenyl, benzyl;
R3=C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl, R5COxe2x80x94, R5SO2xe2x80x94, R5OC(O)xe2x80x94, R5NHCOxe2x80x94;
R4=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R5=Ar-0-6alkyl, Het-C0-6alkyl;
R6=H, C1-6alkyl, CH2CF3, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R7=C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R8=H; C2-6 alkenyl; C2-6alkynyl; Het; Ar; C1-6alkyl, optionally substituted by ORxe2x80x2, SRxe2x80x2, NRxe2x80x22, CO2Rxe2x80x2, CO2NRxe2x80x22, N(Cxe2x95x90NH)NH2, Het or Ar;
R9=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R10=C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R11=H, C1-6alkyl, Arxe2x80x94C1-6alkyl, Het-C0-6alkyl, or 
R12=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R13=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl; 
R15=H, C1-6alkyl, C2-6alkeny), C2-6alkynyl, Ar, Het, or C1-6alkyl optionally substituted by OR9, NR92, CONR92, N(Cxe2x95x90NH)NHxe2x80x94, Het or Ar;
R16=C2-6alkyl, C2-6alkenyl, C2-6alkynyl, Ar, Het, or C2-6alkyl optionally substituted by OR9, SR9, NR92, CO2R9, CONR92, N(Cxe2x95x90NH)NHxe2x80x94, Het or Ar;
R19=H, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, Ar, Het, or C1-6alkyl optionally substituted by OR9, SR9, NR92, CO2R9, CONR92, N(Cxe2x95x90NH)NHxe2x80x94, Het or Ar;
R17=R72=H, C1-6alkyl, R10, R10C(O)xe2x80x94, R10C(S)xe2x80x94, R10OC(O)xe2x80x94;
R21=R26=C5-6alkyl; C2-6alkenyl; C3-11cycloalkyl; Txe2x80x94C3-6alkyl; Vxe2x80x94C1-6alkyl; Txe2x80x94C2-6alkenyl; Txe2x80x94(CH2)nCH(T)(CH2)n; optionally substituted by one or two halogens, SR20, OR20,NR2OR27 or C1-4alkyl;
R27=R28CO, R28OCO;
R28=C1-6alkyl; C3-11cycloalkyl; Ar; Het; Txe2x80x94C1-6alkyl; Txe2x80x94(CH2)nCH(T)(CH2)n; optionally substituted by one or two halogens, SR20, OR20, NR2OR73, C1-6alkyl;
R20=R22=R23=R24=R25=R73=H, C1-4alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl; 
xe2x80x83Cbz-leucinyl-; 2-, 3-, or 4-pyridyl methyloxycarbonyl-leucinyl-; 4-imidazole aceryl-leucinyl-, phenyl acetyl-leucinyl, N,N-dimethyl-glycinyl leucinyl, 4-pyridyl acetyl-leucinyl, 2-pyridyl sulfonyl-leucinyl, 4-pyridyl carbonyl-leucinyl, acetyl-leucinyl, benzoyl-leucinyl, 4-phenoxybenzoyl-, 2- or 3-benzyloxybenzoyl-, biphenyl acetyl, alpha-isobutyl-biphenyl acetyl, Cbz-phenylaaninyl, Cbz-norleucinyl-, Cbz-norvalinyl-, Cbz-glutamyl-, Cbz-epsilon-(t-butyl ester)-glutarnyl; acetyl-leucinyl-, 6- or 8-quinoline carbonyl, biphenyl acetyl, alpha-isobutyl-biphenyl acetyl, acetyl, benzoyl, 2- or 3-benzyloxy benzoyl, 4-phenoxy benzoyl-,
xe2x80x83Cbz-amino acid-; 2-,3-, or 4-pynidylmethyloxyearbonyl-aminoacid-; aryl C0-C6alkyloxy carbonyl-amino acid- , heteroaryl C0-C6alkyloxy carbonyl-amino acid-, aryl C0-C6alkyloxy carbonyl-amino acid-, heteroaryl C0-C6alkyloxy carbonyl-amino acid-, C1-C6alkyloxy carbonyl-amino acid-; C1-C6alkyl carbonyl, aryl C0-C6alkyl carbonyl, heteroaryl C0-C6alkyl carbonyl, aryl C0-C6alkyl carbonyl, heteroaryl C0-C6alkyl carbonyl, C1-C6alkyl sulfonyl, aryl C0-C6alkyl sulfonyl, heteroaryl C0-C6alkyl sulfonyl, aryl C0-C6alkyl sulfonyl, heteroaryl C0-C6alkyl sulfonyl;
R30=xe2x80x94H, C1-6 alkyl; 
xe2x80x83Cbz-leucinyl-; 2-, 3-, or 4-pyridyl methyloxycarbonyl-leucinyl-; 4-imidazole acetyl-leucinyl-, phenyl acetyl-leucinyl, N,N-dimethyl-glycinyl leucinyl, 4-pyridyl acetyl-leucinyl, 2-pyridyl sulfonyl-leucinyl, 4-pyridyl carbonyl-leucinyl, acetyl-leucinyl, benzoyl-leucinyl, 4-phenoxybenzoyl-, 2- or 3-benzyloxybenzoyl-, biphenyl acetyl, alpha-isobutyl-biphenyl acetyl, Cbz-phenylalaninyl, Cbz-norleucinyl-, Cbz-norvalinyl-, Cbz-glutamyl-, Cbz-epsilon-(t-butyl ester)-glutamyl; acetyl-leucinyl-, 6- or 8-quinoline carbonyl, biphenyl acetyl, alpha-isobutyl-biphenyl acetyl, acetyl, benzoyl, 2- or 3-benzyloxy benzoyl, 4-phenoxy benzoyl-,
xe2x80x83Cbz-amino acid-; 2-,3-, or 4-pyridylmethyloxycarbonyl-aminoacid-; aryl C0-C6alkyloxy carbonyl-amino acid-, heteroaryl C0-C6alkyloxy carbonyl-amino acid-, aryl C0-C6alkyloxy carbonyl-amino acid-, heteroaryl C0-C6alkyloxy carbonyl-amino acid-, C1-C6alkyloxy carbonyl-amino acid-; C1-C6alkyl carbonyl, aryl C0-C6alkyl carbonyl, heteroaryl C0-C6alkyl carbonyl, aryl C0-C6alkyl carbonyl, heteroaryl C0-C6alkyl carbonyl, C1-C6alkyl sulfonyl, aryl C0-C6alkyl sulfonyl, heteroaryl C0-C6alkyl sulfonyl, aryl C0-C6alkyl sulfonyl, heteroaryl C0-C6alkyl sulfonyl;
R32OCH2Ar, OCH2C1-6alkyl, aryl substituted C0-6alkyl, heteroaryl substituted C0-6alkyl,4-imidazole methylene; 2-, 3-, or 4-pyridylmethylneneoxy; 4-pyridyl methylene, 2-pyridyl sulfonyl, 4-pyridyl, aryl substituted C0-6alkyloxy, heteroaryl substituted C0-6alkyloxy;
R33=C1-6alkyl, xe2x80x94CH2Ph, xe2x80x94CH2CH2CO2R34;
R34=xe2x80x94H, C1-6alkyl;
R35=Ar, HetAr;
R36=Aryl, heteroaryl, pyridyl, isoquinolinyl;
R37=C1-6alkyl, xe2x80x94CH2Ph, xe2x80x94CH2CH2CO2R34;
R38=Cbz; C1-6alkyl or aryl substituted
xe2x80x83Cbz; C1-6alkyl xe2x80x94CO; benzoyl; C1-6alkyl or aryl substituted benzoyl; 
xe2x80x83Cbz-leucinyl-; 2-, 3-, or 4-pyridyl methyloxycarbonyl-leucinyl-; 4imidazole acetyl-leucinyl-, phenyl acetyl-leucinyl, N,N-dimethyl-glycinyl leucinyl, 4-pyridyl acetyl-leucinyl, 2-pyridyl sulfonyl-leucinyl, 4pyridyl carbonyl-leucinyl, acetyl-leucinyl, benzoyl-leucinyl, 4-phenoxybenzoyl-, 2- or 3-benzyloxybenzoyl-, biphenyl acetyl, alpha-isobutyl-biphenyl acetyl, Cbz-phenylalaninyl, Cbz-norleucinyl-, Cbz-norvalinyl-, Cbz-glutarnyl-, Cbz-epsilon-(t-butyl ester)-glutamyl; acetyl-leucinyl-, 6- or 8-quinoline carbonyl, biphenyl acetyl, alpha- isobutyl-biphenyl acetyl, acetyl, benzoyl, 2- or 3-benzyloxy benzoyl, 4-phenoxy benzoyl-,
xe2x80x83Cbz-amino acid-; 2-,3-, or 4-pyridylmethyloxycarbonyl-aminoacid-; aryl C0-C6alkyloxy carbonyl-amino acid-, heteroaryl C0-C6alkyloxy carbonyl-amino acid-, aryl C0-C6alkyloxy carbonyl-amino acid-,heteroaryl C0-C6alkyloxy carbonyl-amino acid-, C1-C6alkyloxy carbonyl-amino acid-; C1-C6alkyl carbonyl, aryl C0-C6alkyl carbonyl, heteroaryl C0-C6alkyl carbonyl, aryl C0-C6alkyl carbonyl, heteroaryl C0-C6alkyl carbonyl, C1-C6alkyl sulfonyl, aryl C0-C6alkyl sulfonyl, heteroaryl C0-C6alkyl sulfonyl, aryl C0-C6alkyl sulfonyl, heteroaryl C0-C6alkyl sulfonyl;
R40=H and C1-6alkyl;
R41=H and C1-6alkyl;
R42=C1-6alkyl, aryl substituted C1-6alkyl and hetero aryl substituted C1-6alkyl,; H when R43 is C1-6alkyl, aryl substituted C1-6alkyl; and heteroaryl substituted C1-6alkyl;
R43=C1-6alkyl, aryl substituted C1-6alkyl and hetero aryl substituted C1-6alkyl,; H when R42 is C1-6alkyl, aryl substituted C1-6alkyl; and heteroaryl substituted C1-6alkyl;
R44=CH(R53)NR45R54, CH(R55)Ar, C5-6alkyl;
R45=R46=R47=R48=R49=R50=R51=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R52=Ar, Het, CH(R56)Ar, CH(R56)OAr, N(R56)Ar, C1-6alkyl, CH(R56)NR46R57;
R53=C2-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl,
xe2x80x83R53 and R45 may be connected to form a pyrrolidine or piperidine ring;
R54=R57=R47, R47C(O), R47C(S), R47OC(O);
R55=R56=R58=R59=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R60=R61=R62=R63=R64=H, C1-6alkyl, Arxe2x80x94C0-6alkyl, or Het-C0-6alkyl;
R65=C1-6alkyl, Ar, Het, CH(R69)Ar, CH(R69)OAr, N(R69)Ar, CH(R69)NR61R70;
R66=R69=R71=H, C1-6alkyl, (CH2)0-6xe2x80x94C3-6cycloalkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl;
R67=C1-6alkyl, (CH2)0-6xe2x80x94C3-6cycloalkyl, Arxe2x80x94C0-6alkyl, Het-C0-6alkyl; R66 and R67 may be combined to form a 3-7 membered monocyclic or 7-10-membered bicyclic carbocyclic or heterocyclic ring, optionally substituted with 1-4 Of C1-6alkyl, Ph-C0-6alkyl, Het-C0-6alkyl, C1-6alkoxy, Ph-C0-6alkoxy, Het-C0-6alkoxy, OH, (CH2)1-6NR58R59, O(CH2)1-6NR58R59;
R68=R70=R62, R62C(O), R62C(S), R62OC(O), R62OC(O)NR59CH(R71)(CO);
and pharmaceutically acceptable salts thereof.
The compounds of Formula I are hydrazidyl, bis-hydrazidyl and bis-aminomethyl carbonyl compounds having in common key structural features required of protease substrates, most particularly cathepsin K substrates. These structural features endow the present compounds with the appropriate molecular shape necessary to fit into the enzymatic active site, to bind to such active site, and to react with a sulfhydryl group on the active site, thereby blocking the site and inhibiting enzymatic biological activity. Referring to Formula I, such structural features include the central electrophilic carbonyl, a peptidyl or peptidomimetic molecular backbone on either side of the central carbonyl, a terminal carbobenzyloxy moiety (e.g., Cbz-leucinyl), or a mimic thereof, on the backbone on one or both sides of the carbonyl, and optionally, an isobutyl side chain extending from the backbone on one or both sides of the carbonyl.
Compounds of Formula I wherein D=
and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula II.
More preferred embodiments of the present invention include compounds of Formula II wherein:
X=S, Y=CH, and Z=N;
X=CH, Y=S, and Z=N;
X=N, Y=N, and Z=S;
X=N, Y=N, and Z=O; and
X=N, Y=N, and Z=N.
Preferably R1 is H, methyl or isobutyl. Preferably R1 is isobutyl.
Preferably R2 is isobutyl or benzyl.
Preferably R3 is R5OC(O)xe2x80x94, particularly benzyloxycarbonyl.
Preferably A is a D- or L-amino acid or is absent, preferably A is absent.
Preferably W is CN, NHR6, SR6, CONHR6 or CO2R6. Suitably R6 is H, C1-4alkyl, phenyl or benzyl. Typically, W is CO2H, CO2xe2x80x94C1-4alkyl, CO2-Ph, CO2xe2x80x94CH2Ph, CONH2, NH2 or SH.
The following components or formula II are particularly preferred:
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-carboxythiazol-4-yl)-3xe2x80x2-methylbutyl]-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-carboxamidothiazol-4-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-carboethoxythiazol-4-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-cyanothiazol-4-yl)-3xe2x80x2-methylbutyl]-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-[2-(Nxe2x80x2-benzylcarboxamido)thiazol-4-yl]-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-[2-[Nxe2x80x2-(3-methylpropyl)carboxamido]thiazol-4-yl)]-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-[2-[Nxe2x80x2-(2-phenylethyl)carboxamido]thiazol-4yl)]-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4-carboethoxythiazol-2-yl)-3xe2x80x2-methylbutyl]-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4-carboxythiazol-2-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4carboethoxythiadiazol-2-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-carbo-2,2,2-trifluoroethoxythiazol-4-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4-carboetboxyoxadiazol-2-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl-L-leucinyl)amino-N-[1xe2x80x2-(4-carboethoxythiazol-2-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4-carboxamidooxadiazol-2-yl)-3xe2x80x2-methylbutyl]-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-carboethoxythiazol-4-yl)-3xe2x80x2-methylbutyl]-3-phenylpropanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl-L-leucinyl)amino-N-[1xe2x80x2-(2-carboethoxythiazol-4-yl)-3xe2x80x2-methylbutyl]-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(5-mercapto-1,2,4-oxadiazol-3-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-mercaptothiazol-4-yl)-3xe2x80x2-methylbutyl]4-methylpentanamide;
(2S)-2-(benzyloxycarbonyl)amino-N-(4-carboethoxythiazol-2-yl)methyl-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-benzyloxycarbonylthiazol-4-yl)-3xe2x80x2methylbutyl]-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-4-methyl-N-[3xe2x80x2-methyl-1xe2x80x2-(2-phenoxycarbonylthiazol-4-yl)butyl]pentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-4-methyl-N-[3xe2x80x2-methyl-1xe2x80x2-[2-(2-methylpropyloxycarbonyl)thiazol-4-yl]butyl]pentanamide;
(2R,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4-carboethoxythiazol-2-yl)ethyl]-4-methylpentanamide;
(2R,1xe2x80x2R)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4-carboethoxythiazol-2-yl)ethyl]-4-methylpentanamide; and
(2S,1xe2x80x2S)-N-[1xe2x80x2-(2-aminothiazol-4-yl)-3xe2x80x2-methylbutyl]-2-(benzyloxycarbonyl)amino-4-methylpentanamide.
Most particularly preferred compounds of Formula II are:
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(2-carboethoxythiazol-4-yl)-3xe2x80x2-methylbutyl]-4-methylpentanamide;
(2S,1xe2x80x2S)-2-(benzyloxycarbonyl)amino-N-[1xe2x80x2-(4-carboethoxythiazol-2-yl)-3xe2x80x2methylbutyl]4methylpentanamide; and
2S,1xe2x80x2S)-2-(benzyloxycarbonyl-L-leucinyl)amino-N-[1xe2x80x2-(4-carboethoxythiazol-2-yl)-3xe2x80x2-methylbutyl]-4-methylpentanamide.
Compounds of Formula I wherein D=R11Bxe2x80x94 and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula III.
With respect to compounds of Formula m:
Preferably B is 
More preferably B is 
Preferably R11 is 
Preferably R12 and R13 are H.
Preferably R14 is 
Preferably R15, R16, R18 and R19 are C1-6alkyl.
More preferably R15 and R18 are C4-6alkyl.
Preferably Ar is phenyl optionally substituted by one or two groups chosen from halogen, CF3, NO2, SR9, OR9, NR9 or C1-4alkyl.
Preferably R17 and R72 are R10OC(O)xe2x80x94; and more preferably R10 is Arxe2x80x94C1-4alkyl.
Preferably, R16 and R19 are C4-6alkyl; more preferably, R16 and R19 are i-Bu.
Preferably R17 and R72 are Cbz.
One particular embodiment of the invention of Formula III is a compound of Formula F: 
wherein X, Y, Z, R16, R17, R19 and R72 are as described in Formula III.
Most particularly preferred compounds of Formula III are:
(1S)-N-[4[(1-benzyloxycarbonylamino)-3-methylbutyl]thiazol-2-ylcarbonyl]-Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)hydrazide;
N-benzyloxycarbonyl-L-leucinyl-Nxe2x80x2-benzyloxycarbonyl-L-leucinyl-L-leucinylhydrazide; and
(1S)-N-[2-[(1-benzyloxycarbonylamino)-3-methylbutyl]thiazol-4-ylcarbonyl]-Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)hydrazide.
Compounds of Formula I wherein D=
and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula IV.
A more preferred embodiment of the present invention is a compound of Formula IV wherein R21 and R26 are selected from the group consisting of:
N-Cbz-leucinyl, N-Cbz-glycinyl, N-acetyl-leucinyl, N-Cbz-alanyl, 
and R22, R23, R24 and R25 are H.
Particularly preferred embodiments of Formula IV are:
2,2xe2x80x2-(N,Nxe2x80x2-bis-benzyloxycarbonyl-L-leucinyl)carbohydrazide;
2,2xe2x80x2-(N,Nxe2x80x2-bis-cyclohexylacetyl)carbohydrazide;
2,2xe2x80x2-(N,Nxe2x80x2-bis-4-methylpentanoyl)carbohydrazide;
2,2xe2x80x2-(N,Nxe2x80x2-bis-cyclopentylacetyl)carbohydrazide,
2,2xe2x80x2-(N,Nxe2x80x2-bis-benzyloxycarbonylglycinyl)carbohydrazide;
2,2xe2x80x2-(N,Nxe2x80x2-bis-acetyl-L-leucinyl)carbohydrazide;
2,2xe2x80x2-(N,Nxe2x80x2-bis-benzyloxycarbonyl-L-alanyl)carbohydrazide; and
2-(N-benzyloxycarbonyl-L-leucinyl)-2xe2x80x2-[Nxe2x80x2-(4-methylpentanoyl)]carbohydrazide.
2,2xe2x80x2-(N,Nxe2x80x2-bis-benzyloxycarbonyl-L-leucinyl)carbohydrazide is a most preferred embodiment of Formula IV.
Compounds of Formula I wherein D=
and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula V.
In more preferred compounds of Formula V, when R30=C1-C6 alkyl, R30 is preferably Me or xe2x80x94CH2CH2Me2. When R33=C1-C6 alkyl, R33 is preferably xe2x80x94Pr, xe2x80x94Bu, or xe2x80x94CH2CH2Me2. When R34=C1-C6 alkyl, R34 is preferably -t-Bu.
Even more preferred embodiments of Formula V include:
bis-(Cbz-leucinyl)-1,3-diamino-propan-2-one;
bis-1,3-(4-phenoxy-benzoyl)-diamino-propan-2-one;
1-(Cbz-leucinyl)-amino-3-(acetyl-leucinyl)-amino-propan-2-one;
1-(Cbz-leucinyl)-amino-3-(Cbz-glutanyl-t-butyl ester)-amino-propan-2-one;
1-(Cbz-leucinyl)-amino-3-(Cbz-glutamyl)-amino-propan-2-one;
bis-1,3-(Cbz-leucinyl)-diamino-(S)-butanone-2-one;
1-(Cbz-leucinyl)-amino-3-(Cbz-phenylalanyl)-amino-propan-2-one;
1-(Cbz-leucinyl)-amino-3-(Cbz-norleucinyl)-amino-propan-2-one;
1-(Cbz-leucinyl)-amino-3-(Cbz-norvalinyl)-amino-propan-2-one;
bis-1,3-(Cbz-leucinyl)-diamino-5-methyl-(S)-hexan-2one;
1-(acetyl-leucinyl)-amino-3-(4-phenoxy-benzoyl)-amino-propan-2-one:
1-(Cbz-homo-leucinyl)-amino-(Cbz-leucinyl)-3-amino-propan-2-one;
1-(Cbz-leucinyl)-amino-3-(acetyl-leucinyl)-amino-propan-2-one is a most particularly preferred embodiment of the present invention of Formula V.
Compounds of Formula I wherein D=
and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula VI.
More preferably, R35=Ph, 
or pyridine, even more preferably, R35=Ph, 
Ph may be optionally substituted with C1-6alkyl, C1-6alkoxy, halogens and cyano groups. When R35=pyridine, R may be 2-pyridyl, 3-pyridyl, or 4-pyridyl.
Most particularly preferred embodiments of Formula VI include:
bis-1,3-(4-(3-chloro-2-cyano-phenoxy )-phenyl sulfonamido)-propan-2-one;
bis-1,3-(4-phenoxy-phenyl sulfonamido)-propan-2-one.
Compounds of Formula I wherein D=
and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula VII.
More preferably, R36 is selected from the group consisting of: 
and R37=Me in the more preferred compounds of Formula VII.
Particularly preferred embodiments of Formula VII are:
1-(Cbz-leucinyl)-1no-3-(4-(3-chloro-2-cyano-phenoxy)-phenyl sulfonamido)-propan-2-one;
1-(Cbz-feucinyl)-amino-3-(tosyl-amino)-propan-2-one;
1-(Cbz-leucinyl)-amino-3-((4-phenoxy-phenyl)-sulfonamido)-propan-2-one;
1-(Cbz-leucinyl)-amino-3-(2-dibenzofuransulfonamido)-propan-2-one;
1-(Cbz-homo-leucinyl)-amino-3-(2-dibenzofuransulfonamido)-propan-2-one; and
1-(Cbz-leucinyl)-amino-3-(2-dibenzofuransulfonamido)-(S)-butan-2-one.
1-(Cbz-leucinyl)-amino-3-((4-phenoxy-phenyl)-sulfonamido)-propan-2-one, 1-(Cbz-leucinyl)-amino-3-(2-dibenzofuransulfonamido)-propan-2-one, and 1-(Cbz-leucinyl)-amino-3-(2-dibenzofuransulfonamido)-(S)-butan-2-one are most particularly preferred embodiments of Formula VII.
Compounds of Formula I wherein D=
and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula VIII.
A more preferred embodiment of Formula VIII is when R43 is 2-dibenzofuranylsulfonyl.
Particularly preferred embodiments of Formula VIII are:
(S)-Phenylmethyl [1-[[[3-[benzyloxycarbonyl-leucinyl-amino]-2-oxopropyl]-1-(benzyl)amino]carbonyl]-3-methylbutyl]carbamate.
(S)-Phenylmethyl [1-[[[3-[(2-dibenzofuranylsulfonyl)amino]-2-oxopropyl]-3-(benzyl)amino]carbonyl]-3-methylbutyl]carbamate
(S)-Phenylmethyl [1-[[[3-[(2-dibenzofuranylsulfonyl)amino]-2-oxopropyl]-3-(4-pyridinylmethyl)amino]carbonyl]-3-methylbutyl]carbamate
1-[[[3-[(2-dibenzofuranylsulfonyl)amino-2-oxopropyl]-3-(4-pyridinylmethyl) benzamide
(S)-Phenylmethyl [1-[[[3-[(2-dibenzofuranylsulfonyl) amino]-2-oxopropyl]-1-(4-pyridinylmethyl)amino]carbonyl]-3-methylbutyl]carbamate.
(S)-Phenylmethyl [1-[[[3-[(2-dibenzofuranylsulfonyl)amino]-2-oxopropyl]-1-(4-pyridinylmethyl)amino]carbonyl]-3-methylbutyl]carbamate is a most particularly preferred embodiment of Formula VIII.
Compounds of Formula I wherein D=
and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula IX.
Compounds of Formula IX wherein:
R44=CH(R53)NHR54;
R45, R46, R48, R49, R50 and R51 are H;
R47 is independently CH3, benzyl, 2-pyridinylmethoxy, 4-dimethylaminobenzyl;
J=C(O);
R52=Ar, CH(R10)Ar, CH(R10)OAr, N(R10)Ar, CH(R10)NRxe2x80x3R11;
R53=ethyl, i-Bu;
R54=R47, R47C(O), R47OC(O);
R56=H, CH3, i-Bu;
R57=R47, R47OC(O);
Ar=phenyl or naphthyl, optionally substituted by one or more of Ph-C0-6alkyl, Het-C0-6alkyl, C1-6alkoxy, Ph-C0-6alkoxy, Het-C0-6alkoxy, OH, (CH2)1-6NR58R59, O(CH2)1-6NR58R59;
R58, R59=H, C1-6alkyl, Arxe2x80x94C0-6alkyl; Het-C0-6alkyl,
are more preferred embodiments of the present invention.
The following compounds of Formula IX are particularly preferred:
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(4-phenoxyphenylsulfonyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-alanyl)]-2xe2x80x2-[N-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(4-phenylbenzoyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(4-methoxybenzoyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(4-phenoxybenzoyl)]carbohydrazide;
2-(N-acetyl)-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(N-acetyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-alanyl)]carbohydrazide;
2-[N-(N-acetyl-L-alanyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[4-(N,N-dimethylaminomethyl)benzoyl)]]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[4-hydroxy-[3-(4-morpholinomethyl)]]benzoyl]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[4-(N,N-dimethylaminomethyl)benzyloxy]carbonyl-L-leucinyl]carbohydrazide;
2-(N-benzoyl)-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[3-(4-morpholinomethyl)benzoyl]]carbohydrazide;
2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[4-[3-(N-N-dimethylamino)-1-propyloxy]enzoyl]]carbohydrazide;
2-[N-(2-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[3-(4-pyridylmethoxy)benzoyl]]carbohydrazide;
2-[N-(4-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(3-benzyloxy-5-methoxy)benzoyl]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(3-benzyloxy-4,5-dimethoxy)benzoyl]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(3-benzyloxy-5-ethoxy)benzoyl]carbohydrazide;
2-[N-(N-benzyloxycarbonylglycinyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)]carbohydrazide;
2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-L-prolinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(4-phenylphenylacetyl)]carbohydrazide;
(2xe2x80x2S)-2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-2-aminobutyryl)]carbohydrazide;
2,2xe2x80x2-[N,Nxe2x80x2-[bis-(4-phenylphenylacetyl)]]carbohydrazide;
(2xe2x80x2RS)-2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[2-(4-phenylphenoxy)propionyl]carbohydrazide;
2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(4-methylpentanoyl)]carbohydrazide;
(2RS,2xe2x80x2RS)-2,2xe2x80x2-[N,Nxe2x80x2-[bis-[2-(4-phenylphenyl)4 methylpentanoyl)]]]carbohydrazide;
(2xe2x80x2RS)-2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[2-(4-phenylphenyl)-4-methylpentanoyl)]]carbohydrazide;
(2xe2x80x2RS)-2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-[2-(4-phenylphenyl)4-methylpentanoyl)]]carbohydrazide,
2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(N-benzyloxycarbonyl-N-methyl-L-leucinyl)]carbohydrazide;
2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-[N-(2-pyridinylmethoxycarbonyl)-L-leucinyl]]carbohydrazide;
2-[N-[3-(4-pyridylmethoxy)benzoyl]]-2xe2x80x2-[Nxe2x80x2-[N-(2-pyridinylmethoxycarbonyl)-L-leucinyl]]carbohydrazide;
(2RS)-2-[N-[2-(4phenylphenyl)4methylpentanoyl)]]-2xe2x80x2-[Nxe2x80x2-[N-(2-pyridinylmethoxycarbonyl)-L-leucinyl]]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[2-(4-phenylphenyl)4-methylpentanoyl)]]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[2-(4-phenylphenyl)-4-methylpentanoyl)]]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-[N-(4-phenylphenyl)-N-(2-methylpropyl)carbamoyl]]carbohydrazide;
2-[N-(3-benzyloxybenzoyl)]-2xe2x80x2-[Nxe2x80x2-(N-methyl-L-leucinyl)]carbohydrazide;
2-[N-(N-benzyloxycarbonyl-L-leucinyl)]-2xe2x80x2-[Nxe2x80x2-(N-methyl-L-leucinyl)]carbohydrazide.
Compounds of Formula I wherein D=Lxe2x80x94Gxe2x80x94and Q=
are preferred embodiments of the present invention. For the sake of convenience, such compounds are referred to herein after as compounds of Formula X.
With respect to Formula X:
More preferably G is 
More preferably R63 and R64 are H and R66 and R69 are i-butyl.
More preferably R65 is CH(R69)NR61R70, in which R69 is i-butyl and R61 is H. More preferably R70 is R62OC(O), in which R62 is 
Alternately, R65 is Ar or CH(R69)Ar, in which Ar in said R65 group is 
More preferably, L is
CH(R66)NR60R68, CH(R66)Ar, NR66R67, CH(R66)OArxe2x80x2, Ar, or Het, in which R66 is i-butyl and Ar in said L group is 
or Het in said L group is 
More preferably L is NR66R67 or CH(R66)R60R68.
One particularly preferred embodiment is a compound of Formula G: 
Another particularly preferred embodiment is a compound of Formula H: 
The following compounds of Formula X are most particularly preferred:
(1S)-N-[2-[(1-benzyloxycarbonylamino)-3-methylbutyl]thiazol-4-ylcarbonyl]-Nxe2x80x2-(4-phenoxyphenylsulfonyl)hydrazide;
(1S)-N-[4-[1-(N-benzyloxycarbonyl-L-leucinylamino)-3-methylbutyl]thiazol-2-ylcarbonyl]-Nxe2x80x2-(N-benzyloxycarbonyl-L-leucinyl)hydrazide;
(1S)-N-[2-[(1-benzyloxycarbonylamino)-3-methylbutyl]thiazol-4-ylcarbonyl]-Nxe2x80x2-(4-phenylphenylacetyl)hydrazide;
(1S)-N-[2-[(1-benzyloxycarbonylamino)-3-methylbutyl]thiazol-4-ylcarbonyl]-Nxe2x80x2-[3-(4-pryidinylmethoxy)benzoyl]hydrazide;
N-[2-(2-chlorophenoxymethyl)thiazolylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]-Nxe2x80x2-[2-[4-(1,2,3-thiadiazol-4-yl)phenyl]thiazol-4-ylcarbonyl]hydrazide;
N-[2-[3-(4-chlorophenylsulfonylmethyl)thien-2-yl]thiazol-4ylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
(1S,2xe2x80x2RS)-N-[2-[(1-benzyloxycarbonylamino)-3-methylbutyl]thiazol-4-ylcarbonyl]-Nxe2x80x2-[2xe2x80x2-(4-phenylphenylacetyl)4methylpentanoyl]hydrazide;
N-[2-(3-benzyloxyphenyl)thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(2-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
(1RS)-N-[2-[1-(4-phenylphenyl)-3-methylbutyl]thiazolylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-[2-(2-benzyloxyphenyl)thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-[2-[N-methyl-N-(4-phenylphenyl)amino]thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-(N-benzyloxycarbonyl-L-leucinyl)-Nxe2x80x2-[2-(4-phenylbenzyl)thiazol-4-ylcarbonyl]hydrazide;
N-[2-(4-phenylphenylbenzyl)thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)L-leucinyl]hydrazide;
N-(N-benzyloxycarbonyl-L-leucinyl)-Nxe2x80x2-[2-[N-(2-methylpropyl)-N-phenylamino]thiazol-4-ylcarbonyl]hydrazide;
N-[2-[N-(2-methylpropyl)-N-phenylamino]thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-[2-(2-benzyloxyphenyl)thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(3-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-[2-(2-benzyloxyphenyl)thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(2-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-(N-benzyloxycarbonyl-N-methyl-L-leucinyl)-Nxe2x80x2-[2-(2-benzyloxyphenyl)thiazol-4-ylcarbonyl]hydrazide;
N-[2-[N-(2-methylpropyl)-N-phenylamino]thiazol-4ylcarbonyl]-Nxe2x80x2-[N-(2-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide;
N-[2-[N-(2-methylpropyl)-N-phenylamino]thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(3-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide; and
N-[2-(2-methoxyphenyl)thiazol-4-ylcarbonyl]-Nxe2x80x2-[N-(4-pyridinylmethoxycarbonyl)-L-leucinyl]hydrazide.
The present invention includes all hydrates, solvates, complexes and prodrugs of the compounds of this invention. Prodrugs are any covalently bonded compounds which release the active parent drug according to Formula I in vivo. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Inventive compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. In cases in which compounds have unsaturated carbonxe2x80x94carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
The meaning of any substituent at any one occurrence in Formula I or any subformula thereof is independent of its meaning, or any other substituent""s meaning, at any other occurrence, unless specified otherwise.
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of the present invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984). The term xe2x80x9camino acidxe2x80x9d as used herein refers to the D- or L-isomers of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamnic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
xe2x80x9cC1-6alkylxe2x80x9d as applied herein is meant to include substituted and unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. Any C1-6alkyl group may be optionally substituted independently by one or two halogens, SRxe2x80x2, ORxe2x80x2, N(Rxe2x80x2)2, C(O)N(Rxe2x80x2)2, carbamyl or C1-4alkyl, where Rxe2x80x2 is C1-6alkyl. C0alkyl means that no alkyl group is present in the moiety. Thus, Arxe2x80x94C0alkyl is equivalent to Ar.
xe2x80x9cC3-11cycloalkylxe2x80x9d as applied herein is meant to include substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, cycloundecane.
xe2x80x9cC2-6 alkenylxe2x80x9d as applied herein means an alkyl group of 2 to 6 carbons wherein a carbonxe2x80x94carbon single bond is replaced by a carbonxe2x80x94carbon double bond. C2-6alkenyl includes ethylene, 1-propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included.
xe2x80x9cC2-6alkynylxe2x80x9d means an alkyl group of 2 to 6 carbons wherein one carbonxe2x80x94carbon single bond is replaced by a carbonxe2x80x94carbon triple bond. C2-6 alkynyl includes acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne.
xe2x80x9cHalogenxe2x80x9d means F, Cl, Br, and I.
xe2x80x9cArxe2x80x9d or xe2x80x9carylxe2x80x9d means=phenyl or naphthyl, optionally substituted by one or more of Ph-C0-6alkyl, Het-C0-6alkyl, C1-6alkoxy, Ph-C0-6alkoxy, Het-Co -6alkoxy, OH, (CH2)1-6NR58R59, O(CH2)1-6NR58R59; where R58, R59=H, C1-6alkyl; Het-C0-6alkyl, from C1-4alkyl, ORxe2x80x2, N(Rxe2x80x2)2, SRxe2x80x2, CF3, NO2, CN, CO2Rxe2x80x2, CON(Rxe2x80x2), F, Cl, Br and I.
As used herein xe2x80x9cHetxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d represents a stable 5- to 7-membered monocyclic or a stable 7- to 10-membered bicyclic heterocyclic ring, which is either saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure, and may optionally be substituted with one or two moieties selected from C1-4alkyl, ORxe2x80x2, N(Rxe2x80x2)2, SRxe2x80x2, CF3, NO2, CN, CO2Rxe2x80x2, CON(Rxe2x80x2), F, Cl, Br and I, where Rxe2x80x2 is C1-6alkyl. Examples of such heterocycles include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, pyranyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzoxazolyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl.
xe2x80x9cHetArxe2x80x9d or xe2x80x9cheteroarylxe2x80x9d means any heterocyclic moiety encompassed by the above definition of Het which is aromatic in character, e.g., pyridine.
It will be appreciated that the heterocyclic ring described when N=
includes thiazoles, oxazoles, triazoles, thiadiazoles, oxadiazoles, isoxazoles, isothiazols, imidazoles, pyrazines, pyridazines, pyrimidines, triazines and tetrazines which are available by routine chemical synthesis and are stable. The single and double bonds (i.e., ) in such heterocycles are arranged based upon the heteroatoms present so that the heterocycle is aromatic (e.g., it is a heteroaryl group). The term heteroatom as applied herein refers to oxygen, nitrogen and sulfur. When the heteroaryl group comprises a five membered ring, W is preferably an electron withdrawing group, such as halogen, xe2x80x94CN, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94COR7, xe2x80x94CO2R6, xe2x80x94CONHR6, xe2x80x94SO2NHR6, xe2x80x94NHSO2R6, xe2x80x94NHCOR7, xe2x80x94Oxe2x80x94COR6, xe2x80x94SR6 or NRxe2x80x2R6, or a similar electron withdrawing substituent as known in the art.
Certain radical groups are abbreviated herein. t-Bu refers to the tertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical, Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz refers to the benzyloxycarbonyl radical.
Certain reagents are abbreviated herein. DCC refers to dicyclohexylcarbodiimide, DMAP is 2,6-dimethylaminopyridine, EDC refers to N-ethyl-Nxe2x80x2(dimethylaminopropyl)-carbodiimide. HOBT refers to 1-hydroxybenzotriazole, DMF refers to dimethyl formamide, BOP refers to benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate, DMAP is dimethylaminopyridine, Lawesson""s reagent is 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide, NMM is N-methylmorpholine, TFA refers to trifluoroacetic acid, TFAA refers to trifluoroacetic anhydride and THF refers to tetrahydrofuran. Jones reagent is a solution of chromium trioxide, water, and sulfuric acid well-known in the art.
Compounds of Formula II wherein X=CH, Y=S and Z=N, and W=CO2R7 CN, or CONRxe2x80x2R7 may be conveniently prepared by methods analogous to those described in Scheme 1. 
1-Scheme 1 is treated with isobutyl chloroformate and N-methylmorpholine in ethyl acetate to give a mixed anhydride which is treated with diazomethane in ether to provide 2-Scheme 1. The diazoketone is halogenated using 30% HBr in acetic acid in ethyl acetate/ether solution to provide 3-Scheme 1. This material is treated with ethyl thiooxamate in refluxing ethanol to give 4-Scheme 1. The thiazole carboxylic ester is saponified by treatment with a hydroxide base (such as potassium hydroxide, sodium hydroxide or lithium hydroxide) to yield carboxylic acid 5-Scheme 1. The carboxylic acid is treated with isobutyl chloroformate and N-methylmorpholine, followed by gaseous ammonia to provide primary amide 6-Scheme 1 (R3xe2x95x90H). The primary amide is treated with TFAA and pyridine in dichloromethane to provide 7-Scheme 1. Alternatively, 5-Scheme 1 can be converted to substituted amides, 6-Scheme 1, by treatment with alkyl amines (such as benzylamine, 2-phenylethylamine or isobutylamine) and a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chloroformate) in an aprotic solvent (such as dichloromethane or DMF). The carboxylic acid 5-Scheme 1 can be converted to carboxylic esters 8-Scheme 1 by treatment with a primary or secondary alcohol (such as 2,2,2-trifluoroethanol, isobutyl alcohol, benzyl alcohol or phenol) and a dehydrating reagent (such as DCC/DMAP, EDCI or Boc2O/pyridine) in an aprotic solvent (such as dichloromethane or ether). When R2=9-fluorenylmethoxy, treatment of 4-Scheme 1 with piperidine in DMF gives 9-Scheme 1. Treatment of 9-Scheme 1 with a carboxylic acid (such as N-Cbz-L-phenylalanine or N-Cbz-L-leucinyl-L-leucine) and a peptide coupling reagent (such as BOP) in an aprotic solvent (such as dichloromethane) provides 10-Scheme 1. 
Compounds of Formula II wherein X=CH, Y=S and Z=N are prepared by methods analogous to those described in Scheme 1A. 1-Scheme 1A is treated with iodomethane in an aprotic solvent (such as THF) to afford 2-Scheme 1A, which is treated with a primary amine in a protic solvent (such as isopropanol) to give 3-Scheme 1A. this material is then treated with a bromomethyl ketone in a protic solvent (such as ethanol) to provide 4-Scheme 1A. 
Compounds of Formula II wherein X=S, Y=CH and Z=N may be conveniently prepared by methods analogous to those described in Scheme 2. 1-Scheme 2 is treated with isobutyl chloroformate, N-methylmorpholine and ammonia in THF to provide 2-Scheme 2. This material is converted to the thioamide, 3-Scheme 2, by treatment with Lawesson""s reagent in an aprotic solvent (such as THF or toluene). 3-Scheme 2 is converted to the thiazole by condensation with a xcex1-ketoester bearing a suitable leaving group for displacement by a sulfur nucleophile (Cl, Br, I, OMs, O-p-Tos) in dichloromethane. 4-Scheme 2 is treated with TFA to provide 5-Scheme 2. This material is treated with a carboxylic acid (such as N-Cbz-L-leucine, N-Cbz-D-leucine or N-Cbz-L-leucinyl-L-leucine) and a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chloroformate) in an aprotic solvent (such as dichloromethane, DMF or THF) to yield 6-Scheme 2. This material is saponified by treatment with a hydroxide base (such as potassium hydroxide, sodium hydroxide or lithium hydroxide) to yield carboxylic acid 7-Scheme 2. 
Compounds of Formula II wherein X=S, Y=CH and Z=N may also be prepared by methods analogous to those described in Scheme 2A. 1-Scheme 2A is treated with a tert-butoxycarbonyl-protected amino acid (such as N-tert-butoxycarbonyl-L-leucine) and a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chloroformate) in an aprotic solvent (such as dichloromethane, DMF or THF) to yield 2-Scheme 2A, which is treated with trifluoroacetic acid to provide 3-Scheme 2A. This material is treated with a chloroformate (such as 2-biphenylmethyl chloroformate, 2-benzylbenzyl chloroformate, 2-naphthylmethyl chloroformate or 2-phenoxybenzyl chloroformate) and a tertiary amine base (such as diisopropylethylamine) in an aprotic solvent (such as methylene chloride) to provide 4-Scheme 2A. 
Compounds of Formula II wherein X and Y=N, and Z=S may be conveniently prepared by methods analogous to those described in Scheme 3. 1-Scheme 3 is treated with di-tert-butyl dicarbonate and triethylamine in THF to provide 2-Scheme 3. This material is treated with hydrazine hydrate in methanol to provide 3-Scheme 3. The hydrazide is acylated by treatment with ethyl oxalyl chloride and pyridine in dichloromethane to provide 4-Scheme 3. This material is converted to the thiadiazole, 5-Scheme 3, by treatment with Lawesson""s reagent in an aprotic solvent (such as THF or toluene). 5-Scheme 3 is treated with TFA to provide 6-Scheme 3. This material is treated with a carboxylic acid (such as N-Cbz-L-leucine) and a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chloroformate) in an aprotic solvent (such as dichloromethane, DMF or THF) to yield 7-Scheme 3. 
Compounds of Formula II wherein X and Y=N, and Z=O, and W=CO2Et or CONH2 may be conveniently prepared by methods analogous to those described in Scheme 4. 1-Scheme 4 is treated with thionyl chloride and pyridine in ether, followed by refluxing in toluene to provide 2-Scheme 4. The resultant oxadiazole is treated with TFA to provide 3-Scheme 4. This material is treated with a carboxylic acid (such as N-Cbz-L-leucine) and a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chloroformate) in an aprotic solvent (such as dichloromethane, DMF or THF) to yield 4-Scheme 4. The carboxylic ester is treated with ammonia in methanol to yield 5-Scheme 4. 
Compounds of Formula II wherein X and Y=N, and Z=O, and W=SH may be conveniently prepared by methods analogous to those described in Scheme 5. 1-Scheme 5 and 2-Scheme 5 are treated with a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chloroformate) in an aprotic solvent (such as dichloromethane, DMF or THF) to yield 3-Scheme 5. This material is treated with hydrazine hydrate in methanol to provide 4-Scheme 5. Treatment of 4-Scheme 5 with thiophosgene and triethylamine in chloroform provides 5-Scheme 5. 
Compounds of Formula II wherein X=CH, Y=S and Z=N, and W=SH or NH2 may be conveniently prepared by methods analogous to those described in Scheme 6. Condensation of 1-Scheme 6 with ammonium dithiocarbamate in ethanol yielded 2-Scheme 6. Alternatively, 1-Scheme 6 can be condensed with thiourea in ethanol to give 3-Scheme 6. 
Compounds of Formula II wherein X=CH, Y=N and Z=N and W=C may be prepared by methods analogous to those described in Scheme 7. Treatment of 1-Scheme 7 with diethylamine N-oxide should provide 2-Scheme 7. Condensation of 2-Scheme 7 with a 2,3-diaminocarboxylic acid should then provide 3-Scheme 7, which may be converted to a variety of carboxylic acid derivatives using procedures previously described in other schemes.
Compounds of Formula III may be generally prepared by methods common in the art of organic chemistry for coupling carboxylic acid derivatives to hydrazine. Schemes 8, 9 and 10 are illustrative of a method to prepare compounds wherein B or E is a heterocycle. Compounds of Formula X may be conveniently prepared by methods analogous to those described in Schemes 8, 9 and 19-23. 
Compounds wherein X=CH, Y=S and Z=N, are prepared by methods analogous to those described in Scheme 22. 1-Scheme 8 is treated with isobutyl chloroformate and N-methylmorpholine in ether to give a mixed anhydride which is treated with diazomethane in ether to provide 2-Scheme 8. The diazoketone is halogenated using 30% HBr in acetic acid in ether solution to provide 3-Scheme 8. This material is treated with ethyl thiooxamate in refluxing ethanol to give 4-Scheme 8. The thiazole carboxylic ester is treated with a hydrazine (such as hydrazine monohydrate or methyl hydrazine) in ethanol to yield 5-Scheme 8. This material is treated with a carboxylic acid (such as N-Cbz-L-leucine) and a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF) to provide 6-Scheme 8.
Compounds wherein X=S, Y=CH and Z=N, are prepared by methods analogous to those described in Scheme 9. 
1-Scheme 9 is converted to 2-Scheme 9 by treatment with isobutyl chloroformate, N-methylmorpholine and ammonia in THF. 2-Scheme 9 is treated with Lawesson""s reagent in THF to provide the thioamide 3-Scheme 9. This material is converted to the thiazole by condensation with an xcex1-ketoester followed by treatment with trifluoroacetic anhydride and pyridine in methylene chloride to afford 4-Scheme 21 which is converted to 5-Scheme 9 by treatment with hydrazine monohydrate. This material is treated with a sulfonyl chloride (such as 4-phenoxybenzenesulfonyl chloride) and pyridine in an aprotic solvent (such as dichloromethane) to provide 6-Scheme 9. Alternatively, 6-Scheme 9 may be prepared by treatment with a carboxylic acid (such as N-benzyloxycarbonyl-L-leucine, N-benzyloxycarbonyl-N-methyl-L-leucine, N-(2-pyridinylmethoxycarbonyl)-L-leucine, N-(3-pyridinylmethoxycarbonyl)-L-leucine, N-(4-pyridinylmethoxycarbonyl)-L-leucine, 4-biphenylacetic acid, 3-(4-pyridinylmethoxy)benzioc acid, or 4-methyl-2-(4-phenylphenyl)pentanoic acid) and a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF).
Compounds wherein B=
are prepared by routine methods of peptide synthesis as illustrated for instance by Scheme 10. 
Treatment of a mixture of 1-Scheme 10 and 2-Scheme 10 with a peptide coupling reagent (such as BOP or EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF or dichloromethane) provides 3-Scheme 10. This material is treated with hydrazine hydrate in ethanol to yield 4-Scheme 10, which is treated with a carboxylic acid (such as N-Cbz-L-leucine) and a peptide coupling reagent (such as BOP or EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF or dichloromethane) to provide 5-Scheme 10.
Compounds of Formula IV wherein R22, R23, R24 are H, and R21=R26 are prepared by methods analogous to those described in Scheme 11. 
Symmetric compounds of the Formula IX having RCO as the terminal substituent on both sides are prepared by methods analogous to those described in Scheme 11. Treatment of 1-Scheme 11 with a carboxylic acid (such as 4-biphenylacetic acid or 4-methyl-2-(4-phenylphenyl)pentanoic acid) and a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF) provides 2-Scheme 11.
Nonsymmnetric compounds of the Formula IX, and compounds of Formula IV wherein R22, R23, R24 and R25 are H, and R21xe2x89xa0R26, are prepared by methods analogous to those described in Scheme 12. 
Treatment of 1-Scheme 12 with hydrazine hydrate in a protic solvent (such as methanol or ethanol) provides 2-Scheme 12, which is treated phosgene in toluene to afford 3-Scheme 12. This material is treated with hydrazine hydrate in a protic solvent (such as methanol or ethanol) to provide 4-Scheme 12. Treatment of 4-Scheme 12 with a sulfonyl chloride (such as 4-phenoxyphenylsulfonyl chloride), an acid chloride (such as benzoyl chloride), or a carbamoyl chloride (such as N-(2-methylpropyl)-N-(4-phenylphenyl)carbamoyl chloride) and pyridine in DMF affords 5-Scheme-12. Alternatively, 5-Scheme-12 may be prepared by treatment of 4-Scheme 12 with a carboxylic acid (such as N-benzyloxycarbonyl-L-alanine, N-benzyloxycarbonyl-L-proline, N-benzyloxycarbonylglycine, (S)-N-benzyloxycarbonyl-2-aminobutyric acid, N-benzyloxycarbonyl-N-methyl-L-leucine, N-tert-butoxycarbonyl-N-methyl-L-leucine, N-acetyl-L-leucine, N-acetyl-L-alanine, N-(2-pyridinylmethoxycarbonyl)-L-leucine, N-[4-(N,N-dimethylaminomethyl)benzyloxycarbonyl]-L-leucine, 4-phenylbenzoic acid, 4-methoxybenzioc acid, 4-phenoxybenzoic acid, 4-(N,N-dimethylanunomethyl)benzoic acid, 4-hycroxy-3-[N-(4-morpholinomethyl)]benzoic acid, 3-[N-(4-morpholinomethyl)]benzoic acid, 2-benzyloxybenzoic acid, 3-benzyloxybenzoic acid, 4-benzyloxybenzoic acid, 4-(3-dimethylaminomethylpropoxy)benzoic acid, 3-benzyloxy-5-methoxybenzoic acid, 3-benzyloxy-4,5-dimethoxybenzoic acid, 3-benzyloxy-5-ethoxybenzoic acid, 3-(4-pyridinylmethoxy)benzoic acid, 4-biphenylacetic acid, 2-(4-phenylphenoxy)propionic acid or 4-methyl-2-(4-phenylphenyl)pentanoic acid) and a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chioroformate) in an aprotic solvent (such as dichloromethane, DMF or THF). 5-Scheme-12 may also be prepared by treatment of 4-Scheme 12 with an anhydride (such as acetic anhydride). Alternatively, 3 Scheme 12 may be converted directly to 5-Scheme-I by treatment with a hydrazide (such as 4-methylpentanoyl hydrazide or N-methyl-N-benzyloxycarbonyl-L-leucinyl hydrazide). 
Nonsymmetric compounds of Formula IV, wherein R23xe2x89xa0H are prepared by methods analogous to those described in Scheme 12A. 1-Scheme 12A is treated with an aldehyde (such as benzaldehyde) in a protic solvent (such as ethanol) and the resulting imine is treated with borane-THF complex to afford 2-Scheme 12A, which is subsequently treated with phosgene in toluene to afford 3-Scheme 12A. This material is treated with hydrazine hydrate in a protic solvent (such as methanol or ethanol) to provide 4-Scheme 12A. Treatment of 4-Scheme 12A with a carboxylic acid (such as N-benzyloxycarbonyl-L-leucine) and a peptide coupling reagent (such as BOP, EDC.HCl/1-HOBT or N-methylmorpholine/isobutyl chloroformate) in an aprotic solvent (such as dichloromethane, DMF or THF) to yield 5-Scheme-12A.
Compounds of Formulae V-VII may be conveniently prepared by methods analogous to those described in Schemes 13-16. 
1,3-Bis-amido propan-2-ones may be prepared by acylation of 1,3-diamino-propan-2-ol 1-Scheme 13 with a carboxylic acid 2-Scheme 13 or a mixture of 2 different carboxylic acids (2 and 3) in equimolar amounts and a coupling reagent such as a dialkyl carbodiimide such as DCC or EDCI or HBTU/N-methyl morpholine, followed by oxidation of the carbinol to a ketone with an oxidant such as Jones reagent. 
1,3-Bis-sulfonamido propanones may be prepared by sulfonylation of 1,3-diamino-propan-2-ol 1-Scheme 14 with a sulfonyl chloride 2-Scheme 14 and a base such as N-methyl morpholine, followed by oxidation of the carbinol to a ketone with an oxidant such as Jones reagent. 
1-Amido-3-sulfonamido propanones may be prepared by acylation of 1,3-diamino-propan-2-ol 1-Scheme 15 with a carboxylic acid 2-Scheme 15 and a coupling reagent such as a carbodiimide or HBTU/N-methyl morpholine, followed by treatment with an appropriate sulfonyl chloride 3-Scheme 15 and a base such as N-methyl morpholine, followed by oxidation of the carbinol to a ketone with an oxidant such as Jones reagent. 
1-Amido-3-sulfonamido alkan-2-ones that are larger than propan-2-one, such as butan-2-one or 5-methyl-hexan-2-one, can be prepared by converting an N-protected peptide such as Cbz-leu-leu-OH 1-Scheme 16 to its bromo methyl ketone 3-Scheme 16 via a diazo methyl ketone 2-Scheme 16. Then, the bromide 3-Scheme 1 is displaced with sodium azide to give the corresponding azide 4-Scheme 16. Reduction of the carbonyl with a reducing agent such as sodium borohydride gives alcohol 5-Scheme 16. Subsequent reduction of the azide with a reducing agent such as 1,3-propandithiol gives the free amine 6-Scheme 16. Acylation or sulfonylation of the amine gives amide or sulfonamide 7-Scheme 16. Finally, oxidation of the carbinol with an oxidant such as Jones gives the desired compounds.
Compounds of Formula VIII may be conveniently made using methods analogous to those in Schemes 17 and 18. 
Azide opening of glycidol 1-Scheme 17, followed by tosylation of the primary alcohol gave tosylate 2-Scheme 17, which was coupled to Ellman polymer 3-Scheme 17 as described by described in J. Med. Chem. 1995, 38, 1427-1430 to produce polymer 4-Scheme 17, which was reacted with benzyl amine in toluene, then washed extensively with various solvents. Then, the azide was reduced with 1,3-propanedithiol in MeOH, triethylamine, then was washed extensively with various solvents. Coupling of Cbz-leucine 6-Scheme 17 with the diamine 5-Scheme 1l with equimolar amounts and a coupling reagent such as a dialkyl carbodiimide such as DCC or EDCI or HBTU/N-methyl morpholine. Cleavage of the ether linkage to an alcohol was accomplished with trifluoroacetic acid with various scavengers. Finally, oxidation of the carbinol to a ketone 7-Scheme 17 with an oxidant such as Jones reagent provided the desired final product. 
N-(2,3-Epoxypropyl)phthalimide 1-Scheme 18 (Aldrich) was reluxed with an amine such as 4-pyridiyl methyl amine in isopropanol. The secondary amine 2-Scheme 18 was then acylated with an acylating agent such as Cbz leucine or a sulfonylating reagent such as 2-dibenzofuransulfonyl chloride and base such as N-methyl morpholine in DMF. The phthalimide was then removed with hydrazine in MeOH and the resulting free amine was acylated with an acylating agent such as Cbz leucine or a sulfonylating reagent such as 2-dibenzofuransulfonyl chloride and base such as N-methyl morpholine in DMF.
Compounds of Formula IX may conveniently be made using methods analogous to those in Schemes 19 and 20.
Compounds of Formula X may be conveniently made using methods analogous to those described in Schemes 21-27. 
Compounds wherein X=CH, Y=S, Z=N and R4xe2x89xa0H, are prepared by methods analogous to those described in Scheme 19. Carboxylic ester 1-Scheme 19 is treated with a hydroxide base (such as lithoum hydroxide, sodium hydroxide or potassium hydroxide) in methanol/water to provide 2-Scheme 19. 3-Scheme 19 is treated with a hydrazine (such as methylhydrazine) in a protic solvent (such as ethanol) to give 4-Scheme 19. 2-Scheme 12 and 4-Scheme 19 are coupled by treatment with a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF) to provide 5-Scheme 19. 
Compounds wherein X=S, Y=CH, Z=N and V=2-methoxyphenyl or 2-benzyloxyphenyl, are prepared by methods analogous to those described in Scheme 20. Ethyl bromopyruvate (1-Scheme 20) is treated with thiourea in refluxing ethanol to provide 2-Scheme 20, which is treated successively with sodium nitrite and copper (I) bromide in 16% aqueous HBr, and the product was heated in ethanol with a catalytic amount of HBr to give 3-Scheme 20. Treatment of this material with an arylboronic acid (such as 2-benzyloxyphenylboronic acid), tetrakis(triphenylphosphine)palladium(O) and cesium fluoride in refluxing DME provides 4Scheme 20. Alternatively, 4Scheme 20, may be prepared by treatment of 3-Scheme 20 with an arylstannane (such as 2-trimethylstannylanisole) and tetrakis(triphenylphosphine)palladium(O) in refluxing toluene. Treatment of 4 Scheme 20 with hydrazine hydrate in ethanol provides 5-Scheme 20. which is treated with a carboxylic acid (such as N-benzyloxycarbonyl-N-methyl-L-leucine, N-(2-pyridinylmethoxycarbonyl)-L-leucine, N-(3-pyridinylmethoxycarbonyl)-L-leucine or N-(4-pyridinylmethoxycarbonyl)-L-leucine) and a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF) to provide 6-Scheme 20. 
Compounds wherein X=S, Y=CH, Z=N and V=NR66R67, are prepared by methods analogous to those described in Scheme 21. An acid chloride (1-Scheme 21) is treated with a primary amine (such as 4-aminobiphenyl or aniline) and pyridine in an aprotic solvent (such as methylene chloride) to provide 2-Scheme 21, which is treated with lithium aluminum hydride in THF to afford 3-Scheme 25. Treatment of 3-Scheme 21 with thiophosgene and pyridine in methylene chloride, followed by treatment with ammonia in methanol provides 4-Scheme 21. Alternatively, 4-Scheme 21 may be prepared by treatment of 3-Scheme 21 with benzoyl isothiocyanate, followed by treatment of the intermediate benzoyl thiourea with potassium carbonate in methanol/water. 4-Scheme 21 is treated with hydrazine hydrate in ethanol to give 5-Scheme 21. Treatment of 5-Scheme 21 with a carboxylic acid (such as N-(2-pyridinylmethoxycarbonyl)-L-leucine, N-(3-pyridinylmethoxycarbonyl)-L-leucine or N-(4pyridinylmethoxycarbonyl)-L-leucine) and a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF) affords 6-Scheme 21. 
Compounds wherein X and Y=N, and Z=NH, are prepared by methods analogous to those described in Scheme 26. 1-Scheme 22 is treated with hydrazine hydrate in ethanol to give 2-Scheme 22, which is heated with a mixed anhydride to provide triazole 3-Scheme 22. This material is treated with hydrazine hydrate to provide 4-Scheme 22, which is treated with a carboxylic acid (such as N-benzyloxycarbonyl-L-leucine) and a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF) to provide 5-Scheme 22. 
Compounds wherein X=S, Y=CH, Z=N, L=CH(R66)NR60R68 where R68. Boc or Cbz, or R65=CH(R69)NR61R70 where R70xe2x89xa0Boc or Cbz are prepared by methods analogous to those described in Scheme 27. 1-Scheme 23 is treated with trifluoroacetic acid to provide 2-Scheme 23. This material is treated with a carboxylic acid (such as pryazinecarboxylic acid, isonicotinic acid, 4-imidazolylacetic acid or pipecolic acid) and a peptide coupling reagent (such as EDC.HCl/1-HOBT) in an aprotic solvent (such as DMF) to provide 3-Scheme 23. 3-Scheme 23 may also be prepared by treatment of 2-Scheme 23 with a sulfonyl chloride (such as 2-pyridinesulfonyl chloride) and a tertieary amine base (such as diisopropylethylamine) in an aprotic solvent (such as methylene chloride). Alternatively, treatment of 4-Scheme 23 with trifluoroacetic acid provides 5 Scheme 23. 
1,3-Diamino-propan-2-ol (or an N-alkyl substituted diamino-propanol) is coupled to a protected leucine analog (either Cbz- or Boc-) and another carboxylic acid or sulfonyl chloride. Removal of the protective group, followed by acylation or sulfonylation, and oxidation of the alcohol provides the desired compounds. 
N-Allyl amine (or a N-alkyl-N-allyl amine) is coupled to a Cbz-amino acid (or sulfonylated with an aryl sulfonyl chloride), then the alkene is epoxidized with a peracid (or dimethyl diooxirane). The epoxide is opened with a subsituted amine, then the amine is acylated or sulfonylated. Final oxidation gives the desired ketones.
The starting materials used herein are commercially available amino acids or are prepared by routine methods well known to those of ordinary skill in the art and can be found in standard reference books, such as the COMPENDIUM OF ORGANIC SYNTHETIC METHODS, Vol. I-VI (published by Wiley-Interscience).
Coupling methods to form amide bonds herein are generally well known to the art. The methods of peptide synthesis generally set forth by Bodansky et al., THE PRACTICE OF PEPTIDE SYNTHESIS, Springer-Verlag, Berlin, 1984; E. Gross and J. Meienhofer, THE PEPTIDES, Vol. 1, 1-284 (1979); and J. M. Stewart and J. D. Young, SOLID PHASE PEPTIDE SYNTHESIS, 2d Ed., Pierce Chemical Co., Rockford, Ill., 1984. are generally illustrative of the technique and are incorporated herein by reference.
Synthetic methods to prepare the compounds of this invention frequently employ protective groups to mask a reactive functionality or minimize unwanted side reactions. Such protective groups are described generally in Green, T. W, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley and Sons, New York (1981). The term xe2x80x9camino protecting groupsxe2x80x9d generally refers to the Boc, acetyl, benzoyl, Fmoc and Cbz groups and derivatives thereof as known to the art. Methods for protection and deprotection, and replacement of an amino protecting group with another moiety are well known.
Acid addition salts of the compounds of Formula I are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts. Halides, sulfate, phosphate, alkanoates (such as acetate and trifluoroacetate), benzoates, and sulfonates (such as mesylate) are examples of anions present in pharmaceutically acceptable salts.
This invention also provides a pharmaceutical composition which comprises a compound according to Formula I and a pharmaceutically acceptable carrier, diluent or excipient. Accordingly, the compounds of Formula I may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of Formula I prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
The compounds of Formula I are useful as protease inhibitors, particularly as inhibitors of cysteine and serine proteases, more particularly as inhibitors of cysteine proteases, even more particularly as inhibitors of cysteine proteases of the papain superfanmily, yet more particularly as inhibitors of cysteine proteases of the cathepsin family, most particularly as inhibitors of cathepsin K. The present invention also provides useful compositions and formulations of said compounds, including pharmaceutical compositions and formulations of said compounds.
The present compounds are useful for treating diseases in which cysteine proteases are implicated, including infections by pneumocystis care, trypsanoma cruzi, trypsanoma brucei, and Crithidia fusiculata; as well as in schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy; and especially diseases in which cathepsin K is implicated, most particularly diseases of excessive bone or cartilage loss, including osteoporosis, gingival disease including gingivitis and periodontitis, arthritis, more specifically, osteoarthritis and rheumatoid arthritis, Paget""s disease; hypercalcemia of malignancy, and metabolic bone disease.
Metastatic neoplastic cells also typically express high levels of proteolytic enzymes that degrade the surrounding matrix, and certain tumors and metastatic neoplasias may be effectively treated with the compounds of this invention.
The present invention also provides methods of treatment of diseases caused by pathological levels of proteases, particularly cysteine and serine proteases, more particularly cysteine proteases, even more particularly as inhibitors of cysteine proteases of the papain superfamily, yet more particularly cysteine proteases of the cathepsin family, which methods comprise administering to an animal, particularly a mammal, most particularly a human in need thereof a compound of the present invention. The present invention especially provides methods of treatment of diseases caused by pathological levels of cathepsin K, which methods comprise administering to an animal, particularly a mammal, most particularly a human in need thereof an inhibitor of cathepsin K, including a compound of the present invention. The present invention particularly provides methods for treating diseases in which cysteine proteases are implicated, including infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma brucei, and Crithidia fusiculata; as well as in schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and especially diseases in which cathepsin K is implicated, most particularly diseases of excessive bone or cartilage loss, including osteoporosis-, gingival disease including gingivitis and periodontitis, arthritis, more specifically, osteoarthritis and rheumatoid arthritis, Paget""s disease, hypercalcemia of malignancy, and metabolic bone disease.
This invention further provides a method for treating osteoporosis or inhibiting bone loss which comprises internal administration to a patient of an effective amount of a compound of Formula I, alone or in combination with other inhibitors of bone resorption, such as bisphosphonates (i.e., allendronate), hormone replacement therapy, anti-estrogens, or calcitonin. In addition, treatment with a compound of this invention and an anabolic agent, such as bone morphogenic protein, iproflavone, may be used to prevent bone loss or to increase bone mass.
For acute therapy, parenteral administration of a compound of Formula I is preferred. An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit cathepsin K. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
The compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.
No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.
The compounds of this invention may be tested in one of several biological assays to determine the concentration of compound which is required to have a given pharmacological effect.
Determination of Cathepsin K Proteolytic Catalytic Activity
All assays for cathepsin K were carried out with human recombinant enzyme. Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically Cbz-Phe-Arg-AMC, and were determined in 100 mM Na acetate at pH 5.5 containing 20 mM cysteine and 5 mM EDTA. Stock substrate solutions were prepared at concentrations of 10 or 20 mM in DMSO with 20 uM final substrate concentration in the assays. All assays contained 10% DMSO. Independent experiments found that this level of DMSO had no effect on enzyme activity or kinetic constants. All assays were conducted at ambient temperature. Product fluorescence (excitation at 360 nM; emission at 460 nM) was monitored with a Perceptive Biosystems Cytofluor II fluorescent plate reader. Product progress curves were generated over 20 to 30 minutes following formation of AMC product.
Inhibition Studies
Potential inhibitors were evaluated using the progress curve method. Assays were carried out in the presence of variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of inhibitor and substrate. Data analysis was conducted according to one of two procedures depending on the appearance of the progress curves in the presence of inhibitors. For those compounds whose progress curves were linear, apparent inhibition constants (Ki,app) were calculated according to equation 1 (Brandt et al., Biochemitsry, 1989, 28, 140):
v=VmA/[Ka(1+I/Ki, app)+A]xe2x80x83xe2x80x83(1) 
where v is the velocity of the reaction with maximal velocity Vm, A is the concentration of substrate with Michaelis constant of Ka, and I is the concentration of inhibitor.
For those compounds whose progress curves showed downward curvature characteristic of time-dependent inhibition, the data from individual sets was analyzed to give kobs according to equation 2:
[AMC]=vsst+(v0xe2x88x92vss)[1xe2x88x92exp(xe2x88x92kobst)]/kobsxe2x80x83xe2x80x83(2) 
where [AMC] is the concentration of product formed over time i, v0 is the initial reaction velocity and vss is the final steady state rate. Values for kobs were then analyzed as a linear function of inhibitor concentration to generate an apparent second order rate constant (kobs/inhibitor concentration or kobs/[I]) describing the time-dependent inhibition. A complete discussion of this kinetic treatment has been fully described (Morrison et al., Adv. Enzymol. Relar. Areas Mol. Biol., 1988, 61, 201).
Human Osteoclast Resorption Assay
Aliquots of osteoclastoma-derived cell suspensions were removed from liquid nitrogen storage, warmed rapidly at 37xc2x0 C. and washed xc3x971 in RPMI-1640 medium by centrifugation (1000 rpm, 5 min at 4xc2x0 C.). The medium was aspirated and replaced with murine anti-HLA-DR antibody, diluted 1:3 in RPMI-1640 medium, and incubated for 30 min on ice The cell suspension was mixed frequently.
The cells were washed xc3x972 with cold RPMI-1640 by centrifugation (1000 rpm, 5 min at 4xc2x0 C.) and then transferred to a sterile 15 mL centrifuge tube. The number of mononuclear cells were enumerated in an improved Neubauer counting chamber.
Sufficient magnetic beads (5/mononuclear cell), coated with goat anti-mouse IgG, were removed from their stock bottle and placed into 5 mL of fresh medium (this washes away the toxic azide preservative). The medium was removed by immobilizing the beads on a magnet and is replaced with fresh medium.
The beads were mixed with the cells and the suspension was incubated for 30 min on ice. The suspension was mixed frequently. The bead-coated cells were immobilized on a magnet and the remaining cells (osteoclast-rich fraction) were decanted into a sterile 50 mL centrifuge tube. Fresh medium was added to the bead-coated cells to dislodge any trapped osteoclasts. This wash process was repeated xc3x9710. The bead-coated cells were discarded.
The osteoclasts were enumerated in a counting chamber, using a large-bore disposable plastic pasteur pipette to charge the chamber with the sample. The cells were pelleted by centrifugation and the density of osteoclasts adjusted to 1.5xc3x97104/mL in EMEM medium, supplemented with 10% fetal calf serum and 1.7 g/liter of sodium bicarbonate. 3 mL aliquots of the cell suspension (per treatment) were decanted into 15 mL centrifuge tubes. These cells were pelleted by centrifugation. To each tube 3 mL of the appropriate treatment was added (diluted to 50 uM in the EMEM medium). Also included were appropriate vehicle controls, a positive control (87MEM1 diluted to 100 ug/mL) and an isotype control (IgG2a diluted to 100 ug/mL). The tubes were incubate at 37xc2x0 C. for 30 min.
0.5 mL aliquots of the cells were seeded onto sterile dentine slices in a 48-well plate and incubated at 37xc2x0 C. for 2 h. Each treatment was screened in quadruplicate. The slices were washed in six changes of warm PBS (10 mL/well in a 6-well plate) and then placed into fresh treatment or control and incubated at 37xc2x0 C. for 48 h. The slices were then washed in phosphate buffered saline and fixed in 2% glutaraldehyde (in 0.2M sodium cacodylate) for 5 min., following which they were washed in water and incubated in buffer for 5 min at 37xc2x0 C. The slices were then washed in cold water and incubated in cold acetate buffer/fast red garnet for 5 min at 4xc2x0 C. Excess buffer was aspirated, and the slices were air dried following a wash in water.
The TRAP positive osteoclasts were enumerated by bright-field microscopy and were then removed from the surface of the dentine by sonication. Pit volumes were determined using the Nikon/Lasertec ILM21W confocal microscope.
General
Nuclear magnetic resonance spectra were recorded at either 250 or 400 MHz using, respectively, a Bruker AM 250 or Bruker AC 400 spectrometer. CDCl3 is deuteriochioroform, DMSO-d6 is hexadeuteriodimethylsulfoxide, and CD3OD is tetradeuteriomethanol. Chemical shifts are reported in parts per million (d) downfield from the internal standard tetramethylsilane. Abbreviations for NMR data are as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublets, dt=doublet of triplets, app=apparent, br=broad. J indicates the NMR coupling constant measured in Hertz. Continuous wave infrared (IR) spectra were recorded on a Perkin-Elmer 683 infrared spectrometer, and Fourier transform infrared (FTIR) spectra were recorded on a Nicolet Impact 400 D infrared spectrometer. IR and FTIR spectra were recorded in transmission mode, and band positions are reported in inverse wavenumbers (cmxe2x88x921). Mass spectra were taken on either VG 70 FE, PE Syx API m, or VG ZAB HF instruments, using fast atom bombardment (FAB) or electrospray (ES) ionization techniques. Elemental analyses were obtained using a Perkin-Elmer 240C elemental analyzer. Melting points were taken on a Thomas-Hoover melting point apparatus and are uncorrected. All temperatures are reported in degrees Celsius.
Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates were used for thin layer chromatography. Both flash and gravity chromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh) silica gel.
Where indicated, certain of the materials were purchased from the Aldrich Chemical Co., Milwaukee, Wis., Chemical Dynamics Corp., South Plainfield, N.J., and Advanced Chemtech, Louisville, Ky.